The flavonoids form a large family of low molecular weight polyphenolic compounds, which occur naturally in plant tissues and include the flavonols, flavones, flavanones, catechins, anthocyanins, isoflavonoids, dihydroflavonols and stilbenes (Haslam (1998), Practical Polyphenolics. From Structure to Molecular Recognition and Physiological Action, Cambridge Univ. Press). More than 4000 flavonoids have been described, most are conjugated to sugar molecules and are commonly located in the upper epidermal layers of leaves (Stewart et al., (2000), J. Agric. Food Chem 48:2663–2669).
The prior art provides increasing evidence that flavonoids, especially flavonols are potentially health-protecting components in the human diet. Dietary flavonols help prevent the development of free-radical derived damage to endothelial cells of the coronary arteries and to the development of atherosclerosis (Vinson et al., (1995) J. Agric. Food Chem. 43:2798–2799).
A positive correlation between the high flavonol/flavone intake and a reduction in coronary heart disease has been reported (Hertog et al., (1993) Lancet, 342:1007–1011 and Hertog et al., (1997) Lancet 349:699). A similar relationship was observed in a Finnish cohort study (Knekt et al., (1996) BMJ 312: 478–481) and in the seven countries study (Hertog et al., (1995) Arch Intern Med 155:381–386).
Flavonoids have also been reported to exhibit anti-inflammatory, anti-allergic and vasodilatory activities in vitro (Cook et al., (1996) Nutrit. Biochem. 7:66–76). Such activity has been attributed in part to their ability to act as antioxidants, capable of scavenging free radicals and preventing free radical production.
It is also noteworthy that flavanones possess significant antioxidant activity. For example, naringenin has been reported to exhibit 1.5 times greater antioxidant activity than the vitamins C or E (Rice-Evans et al., (1997) Trends Plant Sci. 2:152–159). Flavanones have also been implicated in cancer prevention. For example, naringenin has been reported to exhibit an anti-oestrogenic activity (Ruh et al., (1995) Biochem. Pharmacol. 50:1485–1493), and also has been shown to inhibit human breast cancer cell proliferation (So et al., (1996) Nutrition and Cancer 26:167–181).
Flavanones and their glycosides are also considered important determinants of taste. For example, in contrast to many other fruit, the genus Citrus is characterised by a substantial accumulation of flavanone glycosides (Berhow and Smolensky, (1995) Plant Sci. 112:139–147). It is noteworthy that in grapefruit the sour taste results mainly from the accumulation of the bitter flavanone glycoside, naringin (Horowitz and Gentili, (1969) J. Agric. Food Chem. 17:696–700). Naringin comprises the flavanone naringenin and a disaccharide neohesperidoside group attached to the 7 position of the A-ring. It is also noteworthy that on a relative scale of bitterness the 7-β-glucoside compounds are between 1/50th to ⅕th as bitter as quinine (Horowitz and Gentili, (1969) J. Agric. Food Chem. 17:696–700).
The potential advantages of providing food products with elevated flavonoid levels are therefore clearly established in the art. It would be desirable to produce plants which intrinsically possess, elevated levels of health protecting compounds such as flavonoids in order to develop food products with enhanced health protecting properties. Traditionally, the approach to improving plant varieties has been based on conventional cross-breeding techniques, however these require time for breeding and growing of successive plant generations. More recently, recombinant DNA technology has been applied to the general problem of modifying plant genomes to produce plants with desired phenotypic traits.
The flavonoid biosynthetic pathway is well established and has been widely studied in a number of different plant species (see, for example, Koes et al., BioEssays, 16, (1994), 123–132). Briefly, three molecules of malonyl-CoA are condensed with one molecule of coumaroyl-CoA, catalysed by the enzyme chalcone synthase, to give naringenin chalcone which rapidly isomerises, catalysed by chalcone isomerase, to naringenin. Subsequent hydroxylation of naringenin, catalysed by flavanone 3-hydroxylase, leads to dihydrokaempferol. Dihydrokaempferol itself can be hydroxylated, catalysed by flavanone 3′-hydroxylase or flavanone 3′,5′-hydroxylase to produce either dihydroquercetin or dihydromyricetin respectively. All three dihydroflavonols subsequently can be converted to anthocyanins (by the action of dihydroflavonol reductase and flavonoid glucosyltransferase) or alternatively converted to flavonols such as kaempferol, quercetin and myricetin by the action of flavonol synthase.
W00/04175 Unilever discloses a process for increasing the flavonoid content of plants wherein through over-expression of chalcone isomerase the flavonol content of a tomato plant may be increased in the peel of the tomato fruit.
Furthermore it is suggested that over-expression of a chimeric chalcone synthase gene in the Poplar hybrid Populus tremule×p.alba may give rise to a increase in the level of flavonoid therein. (Niodescu et al, Acta bot. Gallica, 1996, 143(6), 539–546. In one line flavonoids were located in both cortical and peripheral tissues of the stem. In other lines differences in flavonoid were found to be present in superficial tissues.
WO 99/37794 Unilever, discloses a method whereby the incorporation of different transcription factors into the genome of the plant can be used to alter flavonoid levels through the overexpression of genes encoding enzymes of the flavonoid biosynthetic pathway. No reference to overexpression of any specific gene in the pathway is disclosed. Furthermore, the transformed plants provided by this earlier disclosure have been found not to show an increase in both chalcone synthase and flavonol synthase enzyme activities.
The objective technical problem to be solved by the present invention therefore relates to providing an alternative process for increasing the flavonoid content of plants. More-particularly the problem to be solved by the present invention is directed to providing a process which demonstrates enhanced flavonoid synthesis over alternative processes known in the art, specifically in the flesh tissue of fruit.
It was not known at the time of filing that the over-expression of specific combinations of genes which encode enzymes of the flavonoid biosynthetic pathway may be used to increase the content of flavonoids in a plant. Furthermore there was no hint or suggestion in the disclosure of the prior art that any synergistic effect could result from the over-expression of any selected combination of genes encoding enzymes of the flavonoid biosynthetic pathway, nor that such a synergistic effect could be localised to particular edible tissues of a plant. In particular it was not known at the time of filing that increasing the activities of chalcone synthase and flavonol synthase could bring about such results.
Definition of Terms
The expression “plant” is used to refer to a whole plant or part thereof, a plant cell or group of plant cells. Preferably however the invention is particularly directed at transforming whole plants and the use of the whole plant or significant parts thereof such as leaves, fruit, seeds or tubers.
A “pericarp tissue” is used to refer to the wall of a fruit, developing from the ovary wall after fertilisation has occurred.
A “columella tissue” is used to refer to the central axis of a fruit, including placental tissue.
A “flavonoid” or a “flavanone” or a “flavonol” may suitably be an aglycone or a conjugate thereof, such as a glycoside, or a methly, acyl or sulfate derivate. Moreover quercitin and kaempferol as used herein can be taken to refer to either the aglycon or glycoside forms, wherein analysis of the non-hydrolysed extract achieves the glycoside, whereas analysis of the hydrolysed extract achieves the aglycon.
A “gene” is a DNA sequence encoding a protein, including modified or synthetic DNA sequences or naturally occurring sequences encoding a protein, and excluding the 5′ sequence which drives the initiation of transcription.
A “DNA sequence functionally equivalent thereto” is any sequence which encodes a protein which has similar functional properties.
According to another embodiment, a functionally equivalent DNA sequence shows at least 50% identity to the respective DNA sequence. More preferably a functionally equivalent DNA sequence shows at least 60%, more preferred at least 75%, even more preferred at least 80%, even more preferred at least 90%, most preferred 95–100% identity, to the respective DNA sequence.
According to the most preferred embodiment a functionally equivalent DNA sequence shows not more than 5 base pairs difference to the respective DNA sequence, more preferred less than 3, e.g. only 1 or 2 base pairs different.
According to another embodiment a functionally equivalent sequence is preferably capable of hybridising under low stringent (2×SSC, 0.1% SDS at 25° C. for 20 min, Sambrook et al 1986) conditions to the respective sequence.
Preferably an equivalent DNA sequence is capable of transcription and subsequent translation to an amino acid sequence showing at least 50% identity to the amino acid sequence encoded by the respective DNA sequence (DNAStar MegAlign Software Version 4.05 and the Clustal algorithm set to default parameters). More preferred, the amino acid sequence translated from an equivalent DNA sequence translated from an equivalent DNA sequence has at least 60%, more preferred at least 75%, even more preferred at least 80%, even more preferred at least 90%, most preferred 95–100% identity to the amino acid sequence encoded by the respective DNA sequence.
“Breaker” is the ripening stage corresponding to the appearance of the first flush of colour on the green fruit.
“Operably linked to a promoter” means the gene, or DNA sequence, is positioned or connected to a promoter in such a way to ensure its functioning. The promoter is any sequence sufficient to allow the DNA to be transcribed. After the gene and promoter sequences are joined, upon activation of the promoter, the gene will be expressed.
A “construct” is a polynucleotide comprising nucleotide sequences not normally associated with nature.
An “increased” level of flavonoids is used to indicate that the level of flavonoids is preferably at least 4 times higher than in similar untransformed plants, more preferred 5–10, most preferred 10–40 times higher than in similar untransformed plants.